The invention relates to a process for enzymatically oxidizing halogenated aromatic compounds.
Chlorinated aromatic compounds such as the chlorobenzene and polychlorinated biphenyls (PCBs) are among the most wide-spread organic contaminants in the environment due to their common application as solvents, biocides, and in the heavy electrical industry. They are also some of the most problematic environmental pollutant, not only because of the health hazards (lipid solubility and hence accumulation in fatty tissues, toxicity and carcinogenicity) but also because of their slow degradation in the environment.
Whilst microorganisms have shown extraordinary abilities to adapt and evolve to degrade most of the organic chemicals released into the environment, the most chemically inert compounds such as PCBs do persist for two main reasons. First, these compounds have very low solubility in water and therefore their bioavailability is low. Research into this problem has focussed on the use of detergents and other surfactants to enhance their solubility and bioavailability. Second, these compounds require activation by enzymatic oxidation or reduction, and it can take a long time for the necessary genetic adaptations by microorganisms to occur, and even then the organisms may not be stable and viable.
We have now found, according to the present invention, that a monoxygenase, in particular P450cam and its physiological electron transfer partners putidaretoxin and putidaretoxin reductase, can be used to oxidise halogenated aromatic compounds. Also mutants of the monoxygenase with substitutions in the active site have enhanced oxidation activity. Thus suitable monoxygenases can be expressed in microorganisms, animals and plants which are going to be used to oxidise the halogenated aromatic compounds.
Accordingly the present invention provides a process for oxidizing a substrate which is a halo aromatic compound, which process comprises oxidizing said substrate with a monooxygenase enzyme.
The process may be carried out in a cell that expresses:
(a) the enzyme
(b) an electron transfer reductase; and
(c) an electron transfer redoxin
The halo aromatic compound is typically a benzene or biphenyl compound. The benzene ring is optionally fused and can be substituted. The halogen is typically chlorine. In many cases there is more than one halogen atom in the molecule, typically 2 to 5 or 6, for example 3. Generally 2 of the halogen atoms will be ortho or para to one another. The compound may or may not contain an oxygen atom such as a hydroxy group, an aryloxy group or a carboxy group. The compound may or may not be chlorophenol or a chlorophenoxyacetic compound.
Specific compounds which can be oxidised by the process of the present invention include 1,2; 1,3- and 1,4-dichlorobenzene, 1,2,4; 1,2,3- and 1,3,5-trichlorobenzene, 1,2,4,5- and 1,2,3,5-tetrachlorobenzene, pentachlorobenzene, hexachlorobenzene, 3,3xe2x80x2-dichlorobiphenyl and 2,3,4,5,6- and 2,2xe2x80x2,4,5,5xe2x80x2-pentachlorobiphenyl.
Other compounds which can be oxidised by the process include recalcitrant halo aromatic compounds, especially dioxins and halogenated dibenzofurans, and the corresponding compounds where one or both oxygen atoms is/are replaced by sulphur, in particular compounds of the formula: 
which possess at least one halo substituent, such as dioxin itself, 2,3,7,8-tetrachlorodibenzioxin.
The oxidation typically gives rise to 1,2 or more oxidation products. These oxidation products will generally comprise 1 or more hydroxyl groups. Generally, therefore, the oxidation products are phenols which can readily be degraded. It is particularly noteworthy that pentachlorobenzene and hexachlorobenzene can be oxidised in this way since they are very difficult to degrade. In contrast the corresponding phenols can be readily degraded by a variety of Pseudomonas and other bacteria. The atom which is oxidized is generally a ring carbon.
The enzyme is typically a natural monooxygenase or a mutant thereof. The natural monooxygenase is generally a prokaryotic or eukaryotic enzyme. Typically it is a haem-containing enzyme and/or a P450 enzyme. The monooxygenase may or may not be a TfdA (2,4-dichlorophenoxy) acetate/xcex1-KG dioxygenase. The monooxygenase is generally of microorganism (e.g. bacterial), fungal, yeast, plant or animal origin, typically of a bacterium of the genus Pseudomonas. These organisms are typically soil, fresh water or salt water dwelling. In the case of a mutant monooxygenase the non-mutant form may or may not be able to oxidize the substrate.
The monooxygenase typically has a coupling efficiency of at least 1%, such as at least 2%, 4%, 6% or more. The monooxygenase typically has a product formation rate of at least 5 minxe2x88x921, such as at least 8, 10, 15, 20, 25, 50, 100, 150 minxe2x88x921 or more. The coupling efficiency or product formation rate is typically measured using any of the substrates or conditions mentioned herein. Thus they are typically measured in the in vitro conditions described in Example 2, in which case the relevant monooxygenase, reductase and redoxin would be present instead of, but at the same concentration as, P450cam, putidaretoxin reductase and putidaretoxin.
The mutant typically has at least one mutation in the active site. A preferred mutant comprises a substitution of an amino acid in the active site by an amino acid with a less polar side chain. Thus the amino acid is typically substituted with an amino acid which is above it in Table 1.
An amino acid xe2x80x98in the active sitexe2x80x99 is one which lines or defines the site in which the substrate is bound during catalysis or one which lines or defines a site through which the substrate must pass before reaching the catalytic site. Therefore such an amino acid typically inateracts with the substrate during entry to the catalytic site or during catalysis. Such an interaction typically occurs through an electrostatic interaction (between charged or polar groups), hydrophobic interaction, hydrogen bonding or van der Waals forces.
The amino acids in the active site can be identified by routine methods to those skilled in the art. These methods include labelling studies in which the enzyme is allowed to bind a substrate which modifies (xe2x80x98labelsxe2x80x99) amino acids which contact the substrate. Alternatively the crystal structure of the enzyme with bound substrate can be obtained in order to deduce the amino acids in the active site.
The monooxygenase typically has 1, 2, 3, 4 or more other mutations, such as substitutions, insertions or deletions. The other mutations may be in the active site or outside the active site. Typically the mutations are in the xe2x80x98second spherexe2x80x99 residues which affect or contact the position or orientation of one or more of the amino acids in the active site. The insertion is typically at the N and/or C terminal and thus the enzyme may be part of a fusion protein. The deletion typically comprises the deletion of amino acids which are not involved in catalysis, such as those outside the active site. The monooxygenase may thus comprise only those amino acids which are required for oxidation activity.
The other mutations in the active site typically alter the position and/or conformation of the substrate when it is bound in the active site. The mutation may make the site on the substrate which is to be oxidized more accessible to the haem group. Thus the mutation may be a substitution to an amino acid which has a smaller or larger, or more or less polar, side chain.
The other mutations typically increase the stability of the protein, or make it easier to purify the protein. They typically prevent the dimerisation of the protein, typically by removing cysteine residues from the protein (e.g. by substitution of cysteine at position 334 of P450cam, or at an equivalent position in a homologue, preferably to alanine). They typically allow the protein to be prepared in soluble form, for example by the introduction of deletions or a poly-histidine tag, or by mutation of the N-terminal membrane anchoring sequence. The mutations typically inhibit protein oligomerisation, such as oligomerisation arising from contacts between hydrophobic patches on protein surfaces.
Typically the mutant monoxygenase is at least 70% homologous to a natural monooxygenase on the basis of amino acid identity.
Any of the homologous proteins mentioned herein are typically at least 70% homologous to a protein or at least 80 or 90% and more preferably at least 95%, 97% or 99% homologous thereto over at least 20, preferably at least 30, for instance at least 40, 60 or 100 or more contiguous amino acids. The contiguous amino acids may include the active site. This homology may alternatively be measured not over contiguous amino acids or nucleotides but over only the amino acids in the active site.
The monoxygenase is preferably:
(i) P450cam,
(ii) a naturally occurring homologue of (i),
(iii) a mutant of (i) or (ii).
Typically (i) is any allelic variant of P450cam of Pseudomonas putida (e.g. of the polypeptide sequence shown in SEQ ID No. 2). Typically (ii) is a species homologue of (i) which has sequence homology with (i), and is typically P450BM-3 of Bacillus megaterium (e.g. the polypeptide sequence shown in SEQ ID No. 4 and the nucelotide sequence is shown in SEQ ID NO: 3), P450terp of Pseudomonas sp, P450eryF of Saccharopollyspora erythraea, or P450 105 D1 (CYP105) of Streptomyces griseus strains.
The active site of (ii) or (iii) may be substantially the same as the active site of (i) or any of the mutants of (i) mentioned herein. Thus the site may comprise the same amino acids in substantially the same positions.
Typically in (iii) amino acid 96 of P450cam, or the equivalent amino acid in a homologue, has been changed to an amino acid with a less polar side chain.
The xe2x80x98equivalentxe2x80x99 side chain in the homologue is one at the homologous position. This can be deduced by lining up the P450cam, sequence and the sequence of the homologue based on the homology between the two sequences. The PILEUP, BLAST and BESTFIT algorithms can be used to line up the sequences (for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990) J Mol Biol 215:403-10 and (Devereux et al (1984) Nucleic Acids Research 12, p387-395)). These algorithms can also be used to calculate the levels of homology discussed herein (for example on their default settings). The equivalent amino acid will generally be in a similar place in the active site of the homologue as amino acid 96 in P450cam.
The discussion below provides examples of the positions at which substitutions may be made in P450cam. The same substitutions may be made at equivalent positions in the homologues. Standard nomenclature is used to denote the mutations. The letter of the amino acid present in the natural form is followed by the position, followed by the amino acid in the mutant. To denote multiple mutations in the same protein each mutation is listed separated by hyphens. The mutations discussed below using this nomenclature specify the natural amino acid in P450cam, but it is to be understood that the mutation could be made to a homologue which has a different amino acid at the equivalent position.
An additional mutation is typically an amino acid substitution at amino acid 87, 98, 101, 185, 244, 247, 248, 296, 395, 396 or a combination of these, for example as shown in table 2.
The following combinations of substitutions are preferred:
(i) Substitution at position 87 to amino acids of different side-chain volume, such as substitutions (typically of F) to A, L, I and W, combined with substitutions at position 96 to amino acids of different side-chain volume such as (typically Y to) A, L, F, and W. These combinations alter the space available in the upper part of the substrate pocket compared to the wild-type enzyme, for example, from Y96W-F87W (little space) to Y96A-F87A (more space), as well as the location of the space, for example from one side in Y96F-F87A to the other in Y96A-F87W.
(ii) Substitution at position 96 to F combined with substitutions at positions 185 and 395. Both T185 and I395 are at the upper part of the substrate pocket, and substitution with A creates more space while substitution with F will reduce the space available and push the substrate close to the haem.
(iii) Substitutions at position 96 to A, L, F, and W combined with substitutions at residues closer to the haem including at 101, 244, 247, 295, 296 and 396 to A, L, F, or W. These combinations will create or reduce space in the region of the different side-chains to offer different binding orientations to substrates of different sizes. For example, the combinations Y96W-L244A and Y96L-V247W will offer very different pockets for the binding of the substrate.
(iv) Triple substitutions at combinations of positions 87, 96, 244, 247, 295, 296, 395 and 396 with combinations of A, L, F, and W. The aim is to vary the size and shape of the hydrophobic substrate binding pocket. For example, the Y96A-F87A-L244A combination creates more space compared to the Y96F-F87W-V396L combination, thus allowing larger substrates to bind to the former while restricting the available binding orientations of smaller substrates in the latter. The combinations Y96F-F87W-V247L and Y96F-F87W-V295I have comparable substrate pocket volumes, but the locations of the space available for substrate binding are very different. The combination Y96F-F87L-V247A has a slightly larger side-chain volume at the 96 position than the combination Y96L-F87L-V247A, but the L side-chain at the 96 position is much more flexible and the substrate binding orientations will be different for the two triple mutants.
(v) The mutants with four or five substitutions were designed with similar principles of manipulating the substrate volume, the different flexibility of various side-chains, and the location of the space available in the substrate pocket for substrate binding so as to effect changes in selectivity of substrate oxidation.
Mutations are generally introduced into the enzyme by using methods known in the art, such as site directed mutagenesis of the enzyme, PCR and gene shuffling methods or by the use of multiple mutagenic oligonucleotides in cycles of site-directed mutagenesis. Thus the mutations may be introduced in a directed or random manner. Typically the mutagenesis method produces one or more polynucleotides encoding one or more different mutants. In one embodiment a library of mutant oligonucleotides is produced which can be used to produce a library of mutant enzymes.
The process is typically carried out in the presence of the natural cofactors of the monooxygenase. Thus typically in addition to the enzyme (a) and the substrate the process is carried out in the presence of an electron transfer reductase (b), an electron transfer redoxin (c), cofactor for the enzyme and an oxygen donor. In this system the flow of electrons is generally: cofactorxe2x86x92(b)xe2x86x92(c)xe2x86x92(a).
(b) is generally an electron transfer reductase which is able to mediate the transfer of electrons from the cofactor to (c), such as a naturally occurring reductase or a protein which has homology with a naturally occurring reductase, such as at least 70% homology, or a fragment of the reductase or homologue. (b) is typically a reductase of any of the organisms mentioned herein, and is typically a flavin dependent reductase, such as putidaredoxin reductase.
(c) is generally an electron transfer redoxin which is able to mediate the transfer of electrons from the cofactor to (a) via (b). (c) is typically a naturally occurring electron transfer redoxin or a protein which has homology with a naturally occuring electron transfer redoxin, such as at least 70% homology; or a fragment of the redoxin or homologue. (c) is typically a redoxin of any of the organisms mentioned herein. (c) is typically a two-iron/two sulphur redoxin, such as putidaredoxin.
The cofactor is any compound capable of donating an electron to (b), such as NADH. The oxygen donor is any compound capable of donating oxygen to (a), such as dioxygen.
Typically (a), (b) and (c) are present as separate proteins; however they may be present in the same fusion protein. Typically only two of them, preferably (b) and (c), are present in the fusion protein. Typically these components are contiguous in the fusion protein and there is no linker peptide present.
Alternatively a linker may be present between the components. The linker generally comprises amino acids that do not have bulky side chains and therefore do not obstruct the folding of the protein subunits. Preferably the amino acids in the linker are uncharged. Preferred amino acids in the linker are glycine, serine, alanine or threonine. In one embodiment the linker comprises the sequence N-Thr-Asp-Gly-Gly-Ser-Ser-Ser-C (SEQ ID NO:6). The linker is typically from at least 5 amino acids long, such as at least 10, 30 or 50 or more amino acids long.
In the process the concentration of (a), (b) or (c) is typically from 10xe2x88x928 to 10xe2x88x922M, preferably from 10xe2x88x926 to 10xe2x88x924M. Typically the ratio of concentrations of (a):(b) and/or (a):(c) is from 0.1:01 to 1:10, preferably from 1:0.5 to 1:2, or from 1:0.8 to 1:1.2. Generally the process is carried out at a temperature and/or pH at which the enzyme is functional, such as when the enzyme has at least 20%, 50%, 80% or more of peak activity. Typically the pH is from 3 to 11, such as 5 to 9 or 6 to 8, preferably 7 to 7.8 or 7.4. Typically the temperature is 10 to 90xc2x0 C., such as 25 to 75xc2x0 C. or 30 to 60xc2x0 C.
In the process different monooxygenases may be present. Typically each of these will be able to oxidise different substrates, and thus using a mixture of monooxygenases will enable a wider range of substrates to be oxidised.
In one embodiment the process is carried out in the presence of a substance able to remove hydrogen peroxide by-product (e.g. a catalase).
In one embodiment the process is carried out in the presence of the enzyme, substrate and an oxygen atom donor, such as hydrogen peroxide or t-butylhydroperoxide, for example using the peroxide shunt.
In one embodiment in the process the (a), (b) and (c) together are typically in a substantially isolated form and/or a substantially purified form, in which case together they will generally comprise at least 90%, e.g., at least 95%, 98% or 99% of the protein in the preparation.
The process may be carried out inside or outside a cell. The cell is typically in culture, at a locus, in vivo or in planta (these aspects are discussed below).
The process is typically carried out at a locus such as in land (e.g in soil) or in water (e.g, fresh water or sea water). When it carried out in culture the culture typically comprises different types of cells of the invention, for example expressing different monooxygenases of the invention. Generally such cells are cultured in the presence of assimible carbon and nitrogen sources.
Typically the cell in which the process is carried out is one in which the monooxygenase does not naturally occur. In another embodiment the monooxygenase is expressed in a cell in which it does naturally occur, but at higher levels than naturally occurring levels. The cell may produce 1, 2, 3, 4 or more different monooxygenases of the invention. These monoxygenases may be capable of oxidizing different halo aromatic compounds. Typically the cell also expresses any of the reductases and/or redoxins discussed above.
The cell is typically produced by introducing into a cell (i.e. transforming the cell with) a vector comprising a polynucleotide that encodes the monooxygenase. The vector may integrate into the genome of the cell or remain extrachromosomal. The cell may develop into the animal or plant discussed below. Typically the coding sequence of the polynucleotide is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell. The control sequence is generally a promoter, typically of the cell in which the monooxygenase expressed.
The term xe2x80x9coperably linkedxe2x80x9d refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence xe2x80x9coperably linkedxe2x80x9d to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
The vector is typically a transposon, plasmid, virus or phage vector. It typically comprises an origin of replication. It typically comprises one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid. The vector is typically introduced into host cells using conventional techniques including calcium phosphate precipitation, DEAE-dextran transfection, or electroporation.
Components (b) and (c) may be expressed in the cell in a similar manner. Typically (a), (b) and (c) are expressed from the same vector, or may be expressed from different vectors. They may be expressed as three different polypeptides. Alternatively they may be expressed in the form of fusion proteins. The cell typically expresses more than one type of monooxygenase.
In one embodiment the three genes encoding the three proteins of the P450cam system, i.e. camA, camB, and camC are placed in the mobile regions of standard transposon vectors and incorporated into the genome of Pseudomonas and flavobacteria. Alternatively plasmid vectors for expressing these genes may used, in which case the P450cam gene cluster will be extra-chromosomal.
The cell may be prokaryotic or eukaryotic and is generally any of the cells or of any of the organisms mentioned herein. Preferred cells are Pseudomanas, flavobacteria or fungi cells (e.g. Aspergillus). In one embodiment the cell is one which in its naturally occurring form is able to oxidise any of the substrates mentioned herein. Typically the cell is in a substantially isolated form and/or substantially purified form, in which case it will generally comprise at least 90%, e.g. at least 95%, 98% or 99% of the cells or dry mass of the preparation.
The invention provides a transgenic animal or plant whose cells are any of the cells of the invention. The animal or plant is transgenic for the monooxygenase gene and typically also an appropriate electron transfer reductase and/or redoxin gene. They may be homozygote or heterozygote for such genes, which are typically transiently introduced into the cells, or stably integrated (e.g. in the genome). The animal is typically a worm (e.g earthworm) or nematode. The plant or animal may be obtained by transforming an appropriate cell (e.g. embryo stem cell, callus or germ cell), fertilising the cell if required, allowing the cell to develop into the animal or plant and breeding the animal or plant true if required. The animal or plant may be obtained by sexual or asexual (e.g cloning) propagation of an animal or plant of the invention or of the F1 organism (or any generation removed from the F1, or the chimera that develops from the transformed cell).
As discussed above the process may be carried out at a locus. Thus the invention also provides a method of treating a locus contaminated with a halo aromatic compound comprising contacting the locus with a monooxygenase, cell, animal or plant of the invention. These organisms are then typically allowed to oxidise the halo aromatic compound. In one embodiment the organisms used to treat the locus are native to the locus. Thus they may be obtained from the locus (e.g. after contamination), transformed/transfected (as discussed above) to express the monooxygenase (and optionally an appropriate electron transfer reductase and/or redoxin.
In one embodiment the locus is treated with more than one type of organism of the invention, e.g. with 2, 3, 4, or more types which express different monooxygenases which oxidise different halo aromatic compounds. In one embodiment such a collection of organisms between them is able to oxidise all halobenzenes, e.g. all chlorobenzenes.
The organisms (e.g. in the form of the collection) may carry out the process of the invention in a bioreactor (e.g. in which they are present in immobilised form). Thus the water or soil to be treated may be passed through such a bioreactor. Soil may be washed with water augmented with surfactants or ethanol and then introduced into the bioreactor.
The invention also provides a process for selecting a mutant of a monooxygenase for its ability to oxidise any of the substrates mentioned herein, which process comprises screening a library of said mutants for their oxidation effect on the substrate. Thus typically the substrate is provided to the library and mutants are selected based on their ability to oxidise the substrate, for example at a particular rate or under particular conditions. The mutant may be selected based on its ability to oxidise the substrate to a particular oxidation product.
Typically the library will be in the form of cells which comprise the mutant enzymes. Generally each cell will express only one particular mutant enzyme. The library typically comprises at least 500 mutants, such as at least 1,000 or 5,000 mutants, preferably at least 10,000 different mutants.
The library typically comprises a random population of mutants. The library may undergo one or more rounds of selection whilst being produced and therefore may not comprise a random population.
The library is typically produced by contacting any of the cells discussed herein which expresses the monooxygenase with a mutagen and/or when the cell is a mutator cell culturing the cell in conditions in which mutants are produced. The mutagen may be contacted with the cell prior to or during culturing of the cell. Thus the mutagen may be present during replication of the cell or replication of the genome of the cell.
The mutagen generally causes random mutations in the polynucleotide sequence which encodes (a). The mutagen is typically a chemical mutagen, such as nitrosomethyguanidine, methyl- or ethylmethane sulphonic acid, nitrite, hydroxylamine, DNA base analogues, and acridine dyes, such as proflavin. It is typically electromagnetic radiation, such as ultra-violet radiation at 260 nm (absorption maximum of DNA) and X-rays. It is typically ionising radiation.
A mutator cell is generally deficient in one or more of the primary DNA repair pathways (such as E. Coli pathways mutS, mutD or mutT, or their equivalents in another organism), and thus has a high mutation rate. Simply culturing such cell leads to the DNA encoding (a) to become mutated. The cell may be of E. Coli XL1 Red mutator strain.
The mutant selected from the library may be used in any aspect of the invention, thus it may be used to oxidise a substrate in the process of the invention or may be expressed in the cell, animal or plant of the invention. It may be used in the method of treating a locus.
The invention is also illustrated by the Examples: